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Combinations of FungalAntagonists for BiologicalControl of Armillaria RootRot of Strawberry PlantsF. Raziq a & R. T.V. Fox ba Department of Plant Pathology , NWFPAgricultural University , Peshawar , Pakistanb Department of Horticulture andLandscape , School of Plant Sciences,University of Reading , 2 Earley Gate,Reading , RG6 6AU , U.K.Published online: 24 Apr 2012.

To cite this article: F. Raziq & R. T.V. Fox (2005) Combinations of FungalAntagonists for Biological Control of Armillaria Root Rot of Strawberry Plants,Biological Agriculture & Horticulture: An International Journal for SustainableProduction Systems, 23:1, 45-57, DOI: 10.1080/01448765.2005.9755307

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Biological Agriculture and Horticulture, 2005, Vol. 23, pp. 45-57 0144-8765/05 $10 © 2005 A B Academic Publishers Printed in Great Britain

Combinations of Fungal Antagonists for Biological Control of Armillaria Root Rot of Strawberry Plants

F. Raziq1* and R.T.V. Fox2

1 Department of Plant Pathology, NWFP Agricultural University, Peshawar, Pakistan. 2Department of Horticulture and Landscape, School of Plant Sciences, University of Reading, 2 Earley Gate, Reading RG6 6AU, U.K.

ABSTRACT

In a pot experiment with strawberry plants (cv. Cambridge Favourite), two isolates (Thl and Th2) of Trichoderma harzianum were tested individually and in combination for their efficacy against the root rot pathogen Armillaria mel/ea. Similarly, an effective isolate of Dactylium dendroides, the 'Shi-itake Pathogen' (SP), was combined with isolates ofT. harzianum, T. viride, T. hamatum and Chaetomium olivaceum for the same purpose. Isolate Th2 performed better alone than in combina­tion with isolate Thl, protecting 75% of the plants from death during the experiment lasting 413 days. None of the plants treated with Thl alone or Thl and Th2 together survived until the end of the experiment. The presence of SP had a significant (p < 0.05) effect on the survival of the plants. The median survival time in the presence of SP was 299.5 days compared with 206.0 days in its absence. At the end of the experiment, which lasted 534 days, 41.7% of the plants treated with SP survived compared with only 16.7% when not treated with the antagonist. The plants also had significantly more living leaves and higher health scores when SP was used. The combination-antagonists did not differ significantly from controls in suppressing the disease and thus enhancing the survival of the plants. The interaction of SP with the combination-antagonists was significant. It improved the efficacy of the other antagonists tested except T. hamatum isolate Tham l and C. olivaceum isolate Co. All the plants treated with T. harzianum isolates Th2 and Th23 or T. viride isolate Tv3 alone died by the end of the experiment, while 50% of them survived when treated with a combination of any of the antagonists and SP. T. hamatum isolate Thaml protected 75% of the plants alone and only 50% in combination with SP.

'Corresponding author: fraziqpk@yahoo.co.uk

45

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46 F. RAZIQ AND R.T.V. FOX

INTRODUCTION

The existing control methods (host resistance, physical and chemical) for infections caused by the species of Armillaria are not practical, effective and reliable (Redfern, 1971; Morrison, 1976; Shaw & Roth, 1978; Hagle & Shaw, 1991; Turner, 1991; West, 1994 ). Results from research investigations have suggested the possibility of successful control with antagonistic organisms, either alone or in integration with another control strategy (Bliss, 1951; Munnecke et al; 1981; Pearce & Malajczuk, 1990; Onsando & Waudo, 1994; Raziq, 1998, 2000; Raziq & Fox, 2003, 2004a, 2004b).

There is a better chance of attaining successful biological control with a mixture of several antagonists than with a single one (Baker & Cook, 1974). Gindrat (1979) also suggested using mixtures of antagonists for biological preparations to achieve a broad spectrum of activity and ecological adaptability. Sivan & Chet (1982) found that an isolate of Trichoderma harzianum was capable of controlling Pythium aphanidermatum better than T. hamatum and at high temperatures, which led them to the idea of using a mixture of several isolates to achieve activity at a wide range of temperature or pH (Sivan et al., 1984 ). Different antagonists may have different modes of action including com­petition, antibiosis and parasitism. They may differ in their response to environmental variations or in their ability to utilize different components of the available substrates. If they interact synergistically, they can control a pathogen more successfully than either of them alone. Such complex associations are also more stable (Baker & Cook, 1974).

The antagonists used in combination should be complementary and not competitive among themselves. They should grow best at different temperatures, water potentials or pH, or on different nutrients so that some will be operative under most environmental conditions (Baker & Cook, 1974).

Trichoderma spp. produce antibiotics such as trichodermin (Ishikawa et al., 1976; Bertagnolli et al., 1998) and 6-pentyl-a-pyrone (6PAP) (Scarselletti & Faull, 1994; Cooney & Lauren, 1998; Pezet et al., 1999). Dactylium dendroides, the 'Shi-itake Pathogen (SP)', was found to be a better competitor in the laboratory and glasshouse studies conducted by Raziq ( 1998). It developed a dense profuse growth on the media plates and J.I. No.2 compost only a few days after application.

Glasshouse experiments were carried out to find out if several combinations of different fungal antagonists could control Armillaria root rot in potted strawberry plants better than either of them alone. One of the experiments was designed to test two isolates of Trichoderma harzianum, while the other one combined isolate SP of D. dendroides with isolates of T. harzianum, T. viride, T. hamatum and Chaetomium olivaceum. Strawberry plants (cv. Cambridge Favourite) were chosen as convenient hosts as they are susceptible to A. mellea, are cheap, easily propagated, and are rapidly infected when inoculated (Fox & Popoola, 1990).

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COMBINING ANTAGONISTS TO CONTROL ARMILLARIA

MATERIALS AND METHODS

Experiment 1: Effect of combining two isolates of Trichoderma harzianum on suppression of Armillaria root rot

47

A glasshouse experiment was carried out to see if the combination of two different isolates of T. harzianum (Th1 and Th2) (Table 1) could control the Armillaria root rot disease more effectively. The strawberry plants were grown in 15 em diameter pots. The pathogen inocula were placed in direct contact with the main roots to ensure quick infection. Only A. me !lea isolate 1 (isolated by the late J. Rishbeth at Cambridge, England, from the roots of a rose plant) was used as this was found the most virulent and fast growing, so the antagonists that could control this isolate would be expected to control the other, less aggressive, isolates as well.

Armillaria mellea isolate I was grown on sterilized hazel billets (5 em long and 1.5-2.0 em in diameter) in glass jars containing 3% malt extract agar (MEA) medium for about 3 weeks at 25°C in the dark. The medium was first inoculated with four 6 mm disks of the isolate cut out with a sterile cork borer from a fresh culture. The inoculum billets were then placed vertically inside the jars on top of the medium. The jars were then sealed with tin foil and incubated.

Some of the treatments in this experiment included living or dead mycelium of Agaricus bisporus mushroom alone to ascertain the possible role of the mushroom mycelium. The antagonists were grown on 50 g mushroom compost, purchased as 5 kg commercial kits from Wyevale Garden Centre, Wokingham, Berkshire, England (manufactured by Harper Mushrooms, Claverdon, Warwickshire), with living mushroom mycelium. In treatments with Th1 or Th2 alone, ten 6 mm diameter colony plugs from 1 week old cultures on 3% MEA medium were added to the mushroom compost in 500 ml glass jars. In the com­bination treatment, five such plugs from each of the two isolates were added. The jars were incubated at 25oC in the dark for 18 days before adding the antagonists

Code

Thl Th2 Th23 Tv3 Thaml Co SP

TABLE I

List of the fungal antagonists used in the glasshouse studies.

Full name

Trichoderma harzianum isolate! T. harzianum isolate 2 T. harzianum isolate 23 T. viride isolate 183289b T. hamatum isolate 321194 Chaetomium olivaceum Dactylium dendroides

Source

J. Fletcher, ADAS, Wye, Kent J. Deacon, University of Edinburgh J. Fletcher, ADAS, Wye, Kent D. Smith, IMI, Egham, Surrey D. Smith, IMI, Egham, Surrey J. Fletcher, ADAS, Wye, Kent A. McQue, The University of Reading

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48 F. RAZIQ AND R.T.V. FOX

to the roots of the strawberry plants and inoculating the plants with Armillaria mellea isolate I. The strawberry plants were inoculated by carefully removing the J.I. No.2 compost used for growing the plants and placing the inoculum billet beside the main root. The antagonists were then added around the roots and the compost replaced. All the treatments were replicated four times in a randomized complete block (RCB) design. The experiment was started on 18 July 1994 at Earley Gate, The University of Reading, U.K., and the strawberry plants were monitored until4 September 1995.

The method of assessment was as follows: the strawberry plants were checked regularly, at least twice a week. The date of death of any plant was recorded and the roots were examined to see if the cause of death was infection by A. me !lea. Infection was assessed visually by splitting the main roots up and looking for the characteristic mycelial sheets in the cambium region. In addition, isolations on artificial media were also attempted. For this purpose, 3% MEA medium was amended with 100 mg I-' of Streptomycin (streptomycin sulphate) and 100 mg 1-1 of Terramycin (oxytetracycline) before pouring into the plates. The roots of the plants still surviving at the end of the experiment were examined for infection and attachment of rhizomorphs. The original inoculum billet was also recovered and its condition noted. The survival data were analysed by Log-Rank test using the lifetest procedure in SAS. A characteristic feature of survival data, which renders standard methods inappropriate, is that survival times are frequently censored. The survival time of an individual is said to be censored when the end­point of interest has not been observed for that individual (Collett, 1994 ). The end-point of interest in these experiments was the time taken by the plants to die of the disease. The !ifetest procedure gave an estimate of the median survival time, the time beyond which 50% of the plants under study were expected to survive. This estimate was, however, not calculated if 50% or more of the plants were censored. Comparisons of the treatments were, therefore, mostly made by the percentage of plants surviving at the end of the experiment. The health scores were recorded on a 0-3 scale where 0 indicated a dead plant and 3 a healthy plant growing vigorously. These data were analysed by ANOV A test.

Experiment 2: Effect of combining Dacty/ium dendroides isolate SP with other antagonists on survival of Armillaria-inoculated strawberry plants

The host plants, A. me !lea isolate used, method of inoculations, and methods of assessments and data analyses were the same as for Experiment 1 above. The fungal antagonists Th2, Th23, Tv3, Thaml, and Co (Table 1) were combined with SP, the 'Shi-itake Pathogen', Dactyli urn dendroides. Each of the antagonists was grown on sterile mushroom compost, obtained from the same source as for

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COMBINING ANTAGONISTS TO CONTROL ARMILLARIA 49

Experiment 1 above, with dead mycelia of Agaricus bisporus. For the treatments of antagonists applied alone, 30 g of the compost was used, while for the combination treatments, 15 g of the compost was used for SP and 15 g for the combination-antagonist, and the two were mixed together before adding to the roots of the strawberry plants which were inoculated with A. me !lea isolate 1 at the same time. The inoculated control treatment had 30 g of the sterile mushroom compost only. All these treatments were replicated four times in an RCB design and the factorial experiment with SP as one factor and combination-antagonists as the other, was started at Earley Gate, The University of Reading, on 8 October 1995. The miticide Pentac was sprayed@ 1 mll-1 on 4 Aprill996 to control red spider mites. The strawberry plants were monitored regularly until 25 March 1997.

RESULTS

Experiment 1: Effect of combining two isolates of Trichoderma harzianum on suppression of Armillaria root rot

Significant (p < 0.05) differences were found among the different treatments (Table 2). Isolate Th2 performed better alone than in combination with isolate Th1, protecting 75% of the plants from death during the experiment lasting 413 days. None of the plants treated with Thl alone or Thl and Th2 combined survived, as was the case with inoculated controls. The strawberry plants treated with the different treatments had no significant differences in mean health scores (Table 3).

Experiment 2: Effect of combining Dactylium dendroides isolate SP with other antagonists on survival of Armillaria-inoculated strawberry plants

The presence of Dactylium dendroides isolate SP had a significant (p < 0.05) effect on the survival of the plants (Table 4). The median survival time in the presence of SP was 299.5 days compared with 206.0 days in its absence. At the end of the experiment, which lasted 534 days, 41.7% of the plants treated with SP survived compared with only 16.7% when not treated with the antagonist. The combination-antagonists did not differ significantly (p = 0.118) from controls in suppressing the disease and thus enhancing the survival of the plants. The interaction of SP with the combination-antagonists was significant (p < 0.05). It improved the efficacy of the other antagonists tested except Trichoderma hamatum isolate Thaml and Chaetomium olivaceum isolate Co. All the plants treated with T. harzianum isolates Th2 and Th23 or T. viride isolate

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50 F. RAZIQ AND R.T.V. FOX

TABLE 2

Survival of Armillaria-inoculated strawberry plants (n = 4) treated with two isolates of Trichoderma harzianum (Th I and Th2) alone and in combination.

Treatment

Healthy control Inoculated control (Armillaria only) 50 g sterilized Agaricus compost 50 g compost with live Agaricus mycelium Thl on 50 g compost with live Agaricus mycelium Th2 on 50 g compost with live Agaricus mycelium Th I and Th2 together on 50 g compost with live Agaricus

mycelium

Log-Rank Test: X2-Value (6 d.f.) = 16.80 (p = 0.01)

Median survival time (days)

314.5 219.0 210.0 194.0

246.5

% Survival

after 413 days

100 0

25 0 0

75

0

- = Figure not available as 50% or more of the plants were censored (plants were censored if they survived at the end of the experiment).

TABLE 3

Effect of combination of two isolates of Trichoderma harzianum (Th I and Th2) on health scores (0-3) of Armillaria-inoculated strawberry plants (n = 4) in the glasshouse after 227 and 252 days.

Health scores

Treatment 227 days 252 days Average

Healthy control 3.00 3.00 3.00 Inoculated control 2.00 2.00 2.00 Sterile compost 0.75 0.75 0.75 Live compost (LC) 1.50 1.00 1.25 Thl on LC 0.75 0.50 0.63 Th2 on LC 2.25 2.25 2.25 Th I and Th2 together on LC 1.50 1.50 1.50

p 0.215 0.082 0.134

Health scores: 0, dead plant: I, plant exhibiting symptoms of active infection; 2, plant with retarded growth; 3, healthy plant growing vigorously.

Tv3 alone died by the end of the experiment while 50% of them survived when treated with a combination of any of the antagonists and SP. Thaml protected 75% of the plants alone and only 50% in combination with SP. Comparable figures for Co were 25% and 0%.

Plants treated with SP had significantly (p < 0.05) more living leaves than those untreated with SP (control) 204 and 297 days after inoculation (Table 5). Significant (p < 0.05) differences were also found among the combination­antagonists. Although none of the combination-antagonists caused a significant

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COMBINING ANTAGONISTS TO CONTROL ARMILLARIA

TABLE 4

Survival of potted strawberry plants protected with combinations of antagonists against Armillaria root rot.

Median survival time (days) (MST) %Survival after 534 days (PS)

51

Antagonist (- SP) (+ SP) Antagonist (-SP) (+ SP) Antagonist

Control 166.5 173.5 0.0 50.0 25.0 Th2 223.0 238.5 0.0 50.0 25.0 Th23 188.0 223.5 0.0 50.0 25.0 Tv3 201.0 251.5 0.0 50.0 25.0 Thaml 75.0 50.0 62.5 Co 184.5 191.0 191.0 25.0 0.0 12.5

MST/PS 206.0 299.5 16.7 41.7

Log-Rank Test:

Category x2 d.f. p

-I+SP (n = 24) 5.64 I 0.018 Antagonist (n = 8) 8.78 5 0.118 -1+ SP x Antagonist (n = 4) 34.36 11 0.000

-1+ SP indicates absence or presence of Dactylium dendroides isolate SP. - = Figure not available as 50% or more of the plants were censored (plants were censored if they survived at the end of the experiment). Antagonists mean the combination-antagonists. Full names of the fungal antagonists are given in Table I.

increase in the mean number of living leaves 115 days after inoculation, after 173 days, it was found that plants treated with Th2 and Tham1 had significantly more leaves than those not treated with any of the combination-antagonists or treated with Tv3 or Co. Only the plants treated with Tham1 maintained significantly higher numbers of living leaves until 297 days after inoculation. The interaction between presence or absence of SP and the combination-antagonists was signifi­cant (p < 0.05) only for the number of leaves counted 204 days after inoculation. Plants treated with Th23 and SP had significantly more leaves than those treated with Th23 alone. The number of leaves on plants treated with any of the other combination-antagonists alone or in combination with SP did not differ significantly.

Plants treated with SP had significantly (p < 0.05) higher mean health scores than those not treated with SP 144 to 329 days after inoculation (Table 6). The combination-antagonists also differed significantly (p < 0.05). One hundred and seventy three days after inoculation, plants treated with Th2 and Thaml had significantly higher health scores than those not treated with any of the combination-antagonists (control). Two hundred and four days after inoculation, plants treated with Th2, Th23, or Tham 1 had significantly higher health scores

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54 F. RAZIQ AND R.T.V. FOX

than those treated with Co or the controls. Thereafter, the differences became non-significant until 329 days after inoculation. The interaction between the presence or absence of SP and the combination-antagonists was significant only for the health scores recorded 204 and 329 days after inoculation. Two hundred and four days after inoculation, plants treated with SP alone had higher mean health scores than those not treated with either SP or any of the combination­antagonists (control). Similarly, plants treated with SP and Th23 had signifi­cantly higher health scores than those treated with Th23 alone. No significant differences were found between the health scores of plants treated with any of the other combination-antagonists and SP or the combination-antagonists alone. Three hundred and twenty nine days after inoculation, plants treated with SP and Th2 had significantly higher health scores than those treated with Th2 alone. No significant differences were found between the health scores of plants treated with any of the other combination-antagonists and SP or the combination­antagonists alone.

DISCUSSION

The results showed that isolate Th2 of Trichoderma harzianum was more effective alone than in combination with isolate Thl. This was because of the inefficiency of Th1, as it alone could not protect the strawberry plants from infection and all the plants died by the end of the experiment lasting 413 days. Th 1 had a more profuse growth on agar media and it perhaps occupied most of the mushroom compost in soil, as it was not colonized adequately by any of the two antagonists at the time of incorporation into the soil compost. This probably restricted the share of the Th2 inoculum and, perhaps, the mixture was predominantly composed of Thl propagules, which failed to protect the straw­berry plants. On the other hand, the whole of the mushroom compost was available to Th2 when this isolate was introduced alone and, because of its superior biocontrol efficacy, it protected 75% of the plants from infection. Larkin & Fravel (1998) tested in various combinations biocontrol isolates that were most effective against Fusarium wilt of tomato in individual assays, but found no better control than that by non-pathogenic Fusarium antagonists alone.

The presence of Dactylium dendroides isolate SP enhanced the survival of the strawberry plants significantly (p < 0.05). This seems to be because of the competitive ability of this antagonist. Following its application to the J.I. No. 2 compost used for growing the strawberry plants, it grew profusely, occupying the whole surface of the compost within a week. It was also found effective against Armillaria in several other glasshouse and field experiments and the in vitro tests (Raziq, 1998). The ability of an antagonist to produce inoculum in excess and to survive, grow, and proliferate in the soil and rhizosphere is con­sidered important for successful biological control (Baker & Cook, 1974; Lewis

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& Papavizas, 1984 ). In addition to these attributes, the biological activities associated with antagonist proliferation are equally, or even more, important (Lewis & Papavizas, 1984). Metabolites produced by SP may also have contributed to its efficacy as a biocontrol agent.

Isolate SP interacted synergistically with T. harzianum isolates Th2 and Th23 and T. vi ride isolate Tv3, resulting in increased survival of the strawberry plants. T. hamatum isolate Tham1 gave a similar level of protection with SP, but was even more effective when applied alone, and Chaetomium olivaceum isolate Co gave poor performance in both cases. Although equal proportions of the plants (50%) were protected by SP alone and in combination with Th2, Th23, Tv3 and Tham1, all the surviving plants treated with SP alone were found infected when the roots were examined, while only half of them were infected when treated with the combinations. This would mean that if the experiment had to continue for a further month or so, all the plants treated with SP alone would have died, particularly because it was the start of the spring, which hastens the death of the infected plants. This signifies the role of the combination antagonists. Trichoderma species are known to display several modes of action against plant pathogens, i.e. antibiosis, competition and parasitism. The synergistic inter­actions are likely to have resulted from additive competition of the antagonists with the pathogen. Active growth of Trichoderma species is also associated with the production of large quantities of extracellular P-(1,3)-glucanase and chitinase, which dissolve pathogen cell walls (Chet et al., 1979).

The superior performance of Tham1 alone may be due to several reasons. Unlike the other Trichoderma isolates, this isolate developed conidia at a later stage and was predominantly in the form of mycelia at the time of application to the compost. Lewis & Papavizas (1984) reported that Trichoderma isolates added to soil as mycelial preparations proliferated more rapidly than conidial preparations. The isolate may have produced more effective antibiotics. T. hamatum has been reported to produce peptide antibiotics different from those produced by the other species (Dennis & Webster, 1971). The antagonist may also have been less affected by the frequent watering in the glasshouse as certain strains of T. hamatum are adapted to conditions of excessive soil moisture (Danielson & Davey, 1973).

The controlled environment of the glasshouse is probably not as good a test of the efficacy of mixtures of antagonists as the natural conditions of the field. Broad spectrum activity and ecological adaptability (Gindrat, 1979), the objectives to be achieved with mixing different isolates, are more of a requirement of the field situations where wide fluctuations over the year in temperature, humidity, pH and microbial interactions are not uncommon. Never­theless, as a first step, glasshouse, and even laboratory, tests should not be overlooked. The results of these glasshouse experiments suggest the possibility of using mixtures of effective antagonists to control Armillaria infections in practical situations, especially those caused by isolates less virulent than A. mellea isolate 1 used in these studies.

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56 F. RAZIQ AND R.T.V. FOX

ACKNOWLEDGEMENT

The authors wish to thank the Government of Pakistan for financial help in the form of a scholarship to the senior author.

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(Received 7 November 2003; accepted 22 August 2004)

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